Gylocosylated humanized B-cell specific antibodies

ABSTRACT

A humanized specific monoclonal antibody or antibody fragment, especially a B-cell specific antibody or antibody fragment, is engineered to contain a glycosylation site in the non-Fc constant region. The glycosylated antibody is useful for diagnosis and/or therapy whenever a targeting antibody or fragment is used, especially for B-cell malignancies. The carbohydrate moiety allows conjugation of labeling or therapeutic agents of increased size, without affecting the binding affinity or specificity of the antibody.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/894,839, filed Jun. 29, 2001, incorporated herein by reference in itsentirety, which is a continuation of U.S. application Ser. No.09/155,107, filed Nov. 17, 1998, incorporated herein by reference in itsentirety, which is a National Stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US97/04196, filed Mar. 19, 1997, whichis an application claiming the benefit under 35 USC 119(e) of U.S.Application Ser. No. 60/013,709, filed Mar. 20, 1996, all of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] The invention relates generally to immunoconjugates fordiagnostic and therapeutic uses in cancer. In particular, the inventionrelates to recombinantly produced humanized monoclonal antibodiesdirected against B-cell lymphoma and leukemia cells, which antibodiescan be covalently conjugated to a diagnostic or therapeutic reagentwithout loss of antibody binding and internalization function and withreduced production of human anti-mouse antibodies.

[0003] Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia areB-cell malignancies that remain important contributors to cancermortality. The response of these malignancies to various forms oftreatment is mixed. They respond reasonably well to chemotherapy, and,in cases where adequate clinical staging of NHL is possible, as forpatients with localized disease, satisfactory treatment may be providedusing field radiation therapy (Hall et al., Radiology for theRadiologist, Lippincott, Philadelphia, 1989, pp 365-376). However, thetoxic side effects associated with chemotherapy and the toxicity to thehematopoietic system from local, as well as whole body, radiotherapy,limits the use of these therapeutic methods. About one-half of thepatients die from the disease (Posner et al., Blood, 61: 705 (1983)).

[0004] The use of targeting monoclonal antibodies conjugated toradionuclides or other cytotoxic agents offers the possibility ofdelivering such agents directly to the tumor site, thereby limiting theexposure of normal tissues to toxic agents (Goldenberg, Semin. Nucl.Med., 19: 332 (1989)). In recent years, the potential of antibody-basedtherapy and its accuracy in the localization of tumor-associatedantigens have been demonstrated both in the laboratory and clinicalstudies (see., e.g., Thorpe, TIBTECH, 11: 42 (1993); Goldenberg,Scientific American, Science & Medicine, 1: 64 (1994); Baldwin et al.,U.S. Pat. Nos. 4,925,922 and 4,916,213; Young, U.S. Pat. No. 4,918,163;U.S. Pat. No. 5,204,095; Irie et al., U.S. Pat. No. 5,196,337; Hellstromet al., U.S. Pat. Nos. 5,134,075 and 5,171,665). In general, the use ofradio-labeled antibodies or antibody fragments against tumor-associatedmarkers for localization of tumors has been more successful than fortherapy, in part because antibody uptake by the tumor is generally low,ranging from only 0.01% to 0.001% of the total dose injected (Vaughan etal., Brit. J. Radiol., 60: 567 (1987)). Increasing the concentration ofthe radiolabel to increase the dosage to the tumor is counterproductivegenerally as this also increases exposure of healthy tissue toradioactivity.

[0005] LL2 (EPB2) is a highly specific anti-B-cell lymphoma andanti-lymphocytic leukemia cell murine monoclonal antibody (mAb) that israpidly internalized by such cells and that can overcome some of theaforementioned difficulties (Shih et al., Int. J. Cancer, 56: 538(1994)). LL2, which is of the IgG2a antibody type, was developed usingthe Raji B-lymphoma cell line as the source of antigen(Pawlak-Byczkowska et al., Cancer Res., 49: 4568 (1989)). Murine LL2(mLL2) is known to react with an epitope of CD22 (Belisle et al., ProcAmer. Assn. Clin. Res., 34: A2873 (1993)). CD22 molecules are expressedin the cytoplasm of progenitor and early pre-B cells, and appear in thecell surface of mature B-cells.

[0006] By immunostaining of tissue sections, mLL2 was shown to reactwith 50 of 51 B-cell lymphomas tested. mLL2 provides a highly sensitivemeans of detecting B-cell lymphoma cell in vivo, as determined by aradioimmunodetection method (Murthy et al., Eur. J. Nucl. Med., 19: 394(1992)). The Fab′ fragment of mLL2 labeled with ^(99m)Tc localized to 63of 65 known lesions in Phase II trial patients with B-cell lymphoma(Mills et al., Proc. Amer. Assn. Cancer Res., 14: A2857 (1993)). Inaddition, ¹³¹I-labeled mLL2 was therapeutically effective in B-celllymphoma patients (Goldenberg et al., J. Clin. Oncol., 9: 548 (1991)).mLL2 Fab′ conjugated to the exotoxin PE38 KDEL induced completeremission of measurable human lymphoma xenografts (CA-46) growing innude mice (Kreitman et al., Cancer Res., 53: 819 (1993)).

[0007] The clinical use of mLL2, just as with most other promisingmurine antibodies, has been limited by the development in humans of ahuman anti mouse antibody response (HAMA). While a HAMA response is notinvariably observed following injection of mLL2, in a significant numberof cases patients developed HAMA following a single treatment with mLL2.This can limit the diagnostic and therapeutic usefulness of suchantibody conjugates, not only because of the potential anaphylacticproblem, but also as a major portion of the circulating conjugate may becomplexed to and sequestered by the circulating anti-mouse antibodies.This is exemplified by one study in which about 30% of the patientsdeveloped low level HAMA response following a single injection of about6 mg of mLL2 ¹³¹I-IgG and nearly all developed a strong HAMA responsewith additional injections. On the other hand, with mLL2 Fab′ labeledwith ^(99m)Tc, no HAMA response was observed. Such HAMA responses ingeneral pose a potential obstacle to realizing the full diagnostic andtherapeutic potential of the mLL2 antibody.

[0008] As noted above, the use of fragments of mLL2, such as F(ab′)₂ andFab′, partially alleviates/circumvents these problems of immunogenicity.However, there are circumstances in which whole IgG is more desirable,such as when induction of cellular immunity is intended for therapy, orwhere an antibody with enhanced survival time is required.

[0009] For monoclonal antibodies to function as the delivery vehiclesfor drugs and radionuclides, it is of prime importance to developmethods for their site-specific conjugations, with minimal perturbationof the resultant immunoreactivities. Most commonly, the conjugation ofdrugs and radionuclides are accomplished through their covalentattachments to side chains of amino acid residues. Due to thenon-site-restricted nature of these residues, it is difficult to avoidundesirable couplings at residues that lie within or are in closevicinity to the ABS, leading to reduced affinity and heterogenousantigen-binding properties. Alternatively, conjugation can be directedat sulfhydryl groups. However, direct labeling relies on the reductionof S—S bonds, with the possible risk of protein fragmentation.

[0010] U.S. patent application Ser. No. 08/289,576, now abandoned, butrefiled as continuation application, U.S. patent application Ser. No.08/690,102, now U.S. Pat. No. 5,789,554, issued on Aug. 4, 1998, theentire disclosure of which is incorporated herein by reference,discloses a humanized mAb having a naturally occurring N-linkedglycosylation site found at amino acid positions 18-20 of the LL2 VKdomain for site-specific drug or chelate conjugation. The attachedcarbohydrate moiety was positioned away from, and demonstrated nophysical contacts with, the antigen binding site (ABS). Theimmunoreactivity of the antibody was not affected when chelates such asDTPA were conjugated to the carbohydrate.

[0011] However, there are limitations to the usefulness of thisantibody. For one, it is not clear what size and type of chelates can beattached before immunoreactivity is affected. We have determined thatattachment of larger chelates does affect the binding affinity. Thus,attachment of an 18 kD Dox-dextran to the carbohydrate at position 18-20of the LL2 VK domain reduces immunoreactivity to about 50%. Furthermore,it would be very advantageous to engineer other antibodies to containactive glycosylation sites. Engineering other antibodies so thatglycosylation sequences are present in the variable region is difficultbecause the engineering steps would need to be repeated for eachantibody. Furthermore, the immunoreactivity of the construct might beaffected.

[0012] IgG glycosylation at Asn-297 in the CH2 Fc domain has beenwell-characterized as important for the maintenance of antibodystability and the appropriate structure for proper effector functions.See Tao and Morrison, J. Immunol. 143: 2595 (1989). Due to therestricted localization of immunoglobulin glycosylation sites, which aredistal to the ABS, oligosaccharide modification of monoclonal antibodieswas used to prepare conjugates. Conjugates modified with ¹³¹I coupled toa tyrosine-containing peptide, which was then site-specifically attachedto oxidized oligosaccharides, exhibited greater targeting efficiencycompared to the conjugates that were modified nonselectively ontyrosine. Because the use of Asn-297-associated carbohydrate requiresthe presence of the Fc portion of the antibody, its use is limited.There are certain applications employing antibody fragments in which theFc portion is not present.

SUMMARY OF THE INVENTION

[0013] The present invention extends those approaches by engineeringN-linked glycosylation sites into the Constant-kappa (CK), a constantlight chain domain and the constant-heavy (CH1) domains. This has thefollowing advantages:

[0014] 1. glycosylation will be on a different domain which isphysically more distant from the variable domains constituting the ABS;

[0015] 2. high dosage conjugation of chelates or even bulky groups whichmight affect the fine structure of the CK or CH1 domain would beexpected to have minimal effects, if any, on the VH and VK domainsforming the ABS;

[0016] 3. antibody fragments, a preferred format in some clinicalapplications, contain both the CH1 and CK domains, and the conjugationsite should be suitable for use in antibody fragments (e.g., Fab,F(ab′)₂);

[0017] 4. unlike the VK-appended glycosylation site which would have tobe introduced (e.g. by site-directed mutagenesis) into differentantibodies on a case-by-case basis, the CK or CH1 domain containing thecarbohydrate addition sites, once identified as an efficient conjugationhandle, can easily be ligated to different variable domains havingdifferent antigen specificities.

[0018] It is an object of this invention to provide humanizedantibodies, having glycosylation in the CK or CH1, domains, that retainantigen binding specificity.

[0019] It is another object of this invention to provide conjugates ofthe glycosylated mAbs containing therapeutic or diagnostic modalities.

[0020] It is a further object of this invention to provide methods oftherapy and diagnosis that utilize the humanized mAbs of the invention.

[0021] In order to achieve these objectives, in one aspect of theinvention, a monoclonal antibody or antibody fragment which isengineered to contain a glycosylation site in the non-Fc constantheavy-chain or light-chain region has been provided. In a preferredembodiment, the monoclonal antibody or antibody fragment is a humanizedantibody or antibody fragment. In another preferred embodiment, thehumanized specific monoclonal antibody is a humanized B-cell specificantibody or antibody fragment. In yet another preferred embodiment, theglycosylation is located on a site selected from the group consisting ofthe HCN1, HCN2, HCN3, HCN4, and HCN5 sites of FIG. 12. In particularlypreferred embodiments, the glycosylation site is the HCN5 site or theHCN1 site of FIG. 12. In a further preferred embodiment, the antibodywhich is engineered to contain a glycosylation site is an antibodyhaving the specificity of the hLL2 antibody.

[0022] In another aspect of the invention, an isolated DNA moleculecomprising an antibody heavy chain gene which comprises a sequencewithin the CH1 region has been provided, which, when the gene iscoexpressed with a second gene for an antibody light chain in a cellsupporting glycosylation, will produce an antibody glycosylated in theCH1 region.

[0023] In a further aspect, an isolated DNA molecule comprising anantibody light chain gene which comprises a sequence within the constantregion has been provided, which, when said gene is coexpressed with asecond gene for an antibody heavy chain in a cell supportingglycosylation, will produce an antibody glycosylated in the constant Kregion.

[0024] In a yet further aspect of the invention, a method of producingan antibody or antibody fragment glycosylated in the constant K and/orCH1 region has been provided comprising coexpressing light and heavychain genes or portions thereof, which have been engineered with amutation such that a glycosylation site is created in the constant Kregion or into the CH1 region of said heavy chain gene or portionsthereof, in a cell that allows glycosylation, such that the antibody orantibody fragment glycosylated in the constant K and/or CH1 region isproduced, and isolating the antibody or antibody fragment.

[0025] In a further still aspect of the invention, a method of diagnosisor treatment of a patient has been provided, wherein a monoclonalantibody or antibody fragment is used to target a specific antigen, theantibody or fragment being used as such or conjugated to a diagnostic ortherapeutic agent,

[0026] the improvement wherein said antibody or fragment is a humanizedmonoclonal antibody or antibody fragment engineered to contain aglycosylation site in the non-Fc constant heavy-chain or light-chainregion. In a preferred embodiment, the antibody or antibody fragment isa B-cell specific antibody or antibody fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows a comparison of murine and humanized LL2 VK (FIG. 1A,SEQ ID NOS: 2, 6 & 20) and VH (FIG. 1B, SEQ ID NOS: 4, 21 & 8) domains.Only hFR sequences (designated as REIHuVK and EUHuVH) different than mFRsequences (designated as murine) are shown, and designated by asterisks.CDRs are boxed. FR residues shown by computer modeling to contact a CDRare underlined.

[0028]FIG. 2 shows the vicinal relationships of LL2 CDRs to theirframework regions (FRs). Separate energy-minimized models for the VL andVH domains of mLL2 were constructed, and all FR residues within a radiusof 4.5 Å or any CDR atom were identified as potential CDR-FR contacts.CDRs of the light (L1, L2, and L3, FIG. 2A) and heavy (H1, H2, and H3,FIG. 2B) chains are shown as “ball and stick” representationssuperimposed on their respective, space-filling FRs.

[0029]FIG. 3A shows the light chain staging (VKpBR) and mammalianexpression (pKH) vectors, and FIG. 3B shows the heavy chain staging(VHpBS) and mammalian expression (pG1g) vectors.

[0030]FIG. 4 shows the double-stranded DNA and amino acid sequences ofthe LL2 VK domain (FIG. 4A, SEQ ID NOS: 1 & 2) and the LL2 VH domain(FIG. 4B, SEQ ID NOS: 3 & 4). Amino acid sequences encoded by thecorresponding DNA sequences are given as one letter codes. CDR aminoacid sequences are boxed. The Asn-glycosylation site located in FR1 ofLL2VK (FIG. 4A) is shown as the underlined NVT sequence.

[0031]FIG. 5A shows the double stranded DNA and corresponding amino acidresidues of the hLL2 VK domain (SEQ ID NOS: 5 & 6). CDR amino acidsequences are boxed. The corresponding data for the VH domain (SEQ IDNOS: 7 & 8) is shown in FIG. 5B.

[0032]FIG. 6 is a schematic diagram representation of the PCR/genesynthesis of the humanized VH region and the subcloning into the stagingvector, VHpBS.

[0033]FIG. 7 shows the results of a comparative Raji cell competitiveantibody binding assay involving mLL2 and cLL2 antibodies competing forbinding to cells against tracer radiolabeled mLL2.

[0034]FIG. 8 shows the results of a comparative Raji cell competitiveantibody binding assay in which mixed humanized/chimeric LL2s werecompared to cLL2 (FIG. 8A), and two versions of hLL2 compared to cLL2(FIG. 8B).

[0035]FIG. 9 shows a comparison of antibody internalization:surfacebinding ratios as a function of time for cLL2, cLL2 (Q to Vmutagenesis), hLL2 and mLL2 antibodies.

[0036]FIG. 10 shows the effect of deglycosylation of mLL2 on its bindingaffinity to Raji cells.

[0037]FIG. 11 shows a competitive binding assay where peroxidaseconjugated mLL2 binding to WN was measured. hLL2 and glycosylatedderivatives in the heavy chain constant regions, at the indicatedconcentrations, were used to compete with mLL2.

[0038]FIG. 12 shows the N-glycan acceptor sequences and positionsintroduced into the CH₁ and CK domains of hLL2 (SEQ ID NOS: 9-19).Site-directed mutagenesis were used to generate the tri-peptide acceptorsequences (shown in bold letters). Partial peptide sequences of the CH₁(H chain) and CK (K chain) domains of hLL2 are shown and alignedaccording to sequence and structure homology to indicate the locationsof engineered potential N-linked glycosylation sites (HCN1—HCN5 andKCN1-KCN4). The β-strand sequences (C—F) are boxed. The residues werenumbered according to Kabat's system; asterisk (*) indicate these heavychain aa residues which were numbered discontinuously from the previousaa residue. The aa residues indicated by * are numbered, from left toright, as 156, 162, 171, 182, 203, and 205, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Glycosylation sites are engineered into CK and CH1 immunoglobulindomains to provide humanized immunoglobulin with engineeredglycosylation sites. By using site-directed mutagenesis, glycosylationsites are engineered into the constant regions of the heavy and lightchains, specifically into the CK and CH1 domains. The mutated CK and CH1nucleotide sequences are then subcloned into light and heavy chainexpression vectors, respectively. The CH1 mutated heavy chain expressionvector is coexpressed with a light chain expression vector to producemutated, humanized antibodies with altered glycosylation sites in theCH1 domain. A similar procedure is followed to produce mutated humanizedantibodies with altered glycosylation sites in the CK domain.

[0040] It should be noted that not all potential carbohydrate-additionsequences can be used for oligosaccharide attachment. A series ofglycosylation mutants were generated by introducing novel N-linkedglycosylation sequences at the heavy chain complementarity determiningregion 2 (CDR2) region of anti-dextran and anti-dansyl antibodies,respectively. While glycosylation as found at Asn 54 and Asn 60 of theanti-dextran antibody, the carbohydrate addition site placed in asimilar position (Asn 55) in the anti-dansyl antibody, however, was notutilized. This “position effect” is not well understood, but is mostlikely to be related to the protein conformation and accessibility ofthe carbohydrate acceptor sequence to glycolyl-transferase.

[0041] In this specification, the expressions “hLL2” or “hLL2 mAb” areintended to refer to the monoclonal antibody constructed by joining orsubcloning the complementarity determining regions (CDRs) of murine VKand VH regions to human framework regions (FRs) and joining orsubcloning these to human constant light and heavy chains, respectively.

[0042] Covalent conjugates between the mutated antibodies of theinvention and a diagnostic or chemotherapeutic reagent, formulated inpharmaceutically acceptable vehicles (see, e.g., Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa.,1990) can be prepared. B cell lymphoma and leukemia specific antibodiescomprising glycosylated CK and CH1 domains conjugated to a diagnostic ortherapeutic reagent resulting in humanized mAbs continue to have theability to internalize into target cells, and to rapidly liberate thediagnostic or chemotherapeutic reagent intracellularly (therebyincreasing effectiveness of the reagent), and the added advantage of areduction of the HAMA response in the human patient.

[0043] Since the carbohydrate moiety of the engineered antibodies of theinvention is not involved in the binding of the antigen, conjugates inwhich a reagent is bound to the antibody through carbohydrate moietiescan be used. For example, a reagent can be conjugated to an oxidizedcarbohydrate derivative. Methods for the production of such conjugates,and their use in diagnostics and therapeutics are provided, for example,in Shih et al., U.S. Pat. No. 5,057,313, Shih et al., Int. J. Cancer 41:832 (1988), and copending, commonly owned Hansen et al., U.S. Ser. No.08/162,912, now U.S. Pat. No. 5,443,953, issued on Aug. 22, 1995, thecontents of which are incorporated herein by reference. Direct linkageof a reagent to oxidized carbohydrate without the use of a polymericcarrier is described in McKearn et al., U.S. Pat. No. 5,156,840, whichis also incorporated by reference.

[0044] A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Theseinclude: chemotherapeutic drugs such as doxorubicin, methotrexate,taxol, and the like; chelator, such as DTPA, to which detectable labelssuch as fluorescent molecules or cytotoxic agents such as heavy metalsor radionuclides can be complexed; and toxins such as Pseudomonasexotoxin, and the like. Several embodiments of these conjugates aredescribed in the examples below.

[0045] Additional or alternative glycosylation sites (NXT/S) can bedesigned and introduced into the Vk, Ck and CH domains of any antibodyaccording to the invention, for example hLL2 (here X stands for anyamino acid but proline or aspartate). The effects on bindingspecificity, biodistribution in vivo, in test animals, and efficiency ofconjugation of drugs and chelates of the glycosylated moieties can beassayed to determine useful glycosylation sites. Likely sites forglycosylation may be identified by comparison with glycosylation sitesfrom known Ab of different species or isotypes, by analysis of the knownstructures of human CK and CH1 domains by computer modeling to identifyexposed positions, or by random shot-gun mutagenesis.

[0046] Cell lines and culture media used in the present inventioninclude LL2 (EPB-2) hybridoma cells (Pawlak-Byczkowska et al. 1989above), Sp2/0-Ag12 myeloma cells (ATCC, Rockville, Md.) and Raji cells.These cells are preferably cultured in Dulbecco's modified Eagle'sMedium (DMEM) supplemented with 10% FCS (Gibco/BRL, Gaithersburg,Mass.), 2 mM L-glutamine and 75 μg/ml gentamicin, (complete DMEM).Transfectomas are grown in Hybridoma Serum Free Medium, HSFM,(Gibco/BRL, Gaithersburg, Mass.) containing 10% of FCS and 75 μg/mlgentamicin (complete HSFM) or, where indicated, in HSFM containing onlyantibiotics. Selection of the transfectomas may be carried out incomplete HSFM containing 500 μg/ml of hygromycin (Calbiochem, San Diego,Calif.). All cell lines are preferably maintained at 37° C. in 5% CO₂.

[0047] Designing Glycosylation Sites in CH1 and CK

[0048] An important aspect of this invention is that antibodyconformations can be modeled by computer modeling (see, for example,Dion, in Goldenberg et al. eds., Cancer Therapy With RadiolabelledAntibodies, CRC Press, Boca Raton, Fla., 1994), which is incorporated byreference. In general, the 3-D structures are best modeled by homology,which is facilitated by the availability of crystallographic data fromthe Protein Data Bank (PDR Code 1REI, Bernstein et al., J. Mol. Biol.112: 535 (1977)), which is incorporated by reference. Similarly, theantibody EU (VH) sequences (Kabat et al., SEQUENCES OF PROTEINS OFIMMUNOLOGICAL INTEREST, 5th edition, US Dept. of Health and HumanServices, US Gov. Printing Office (1991)) can be selected as themodeling counterparts for FR1 to FR3 of the mLL2 heavy chain; FR4 wasbased on NEWM. Id. As X-ray coordinate data is currently lacking for theEU sequence, NEWM structural data (PDR Code 3FAB) for FRs 1 to 4 can beused, and amino acid side groups can be replaced to correspond to mLL2or EU (hLL2) as needed. The CDR of the light chain can be modeled fromthe corresponding sequence of 1MCP Protein Data Bank (L1 and L2) and1REI (L3). For heavy chain CDRs, H1 and H2 can be based on 2HFL ProteinData Bankand 1MCP, respectively, while H3 can be modeled de novo.Wherever possible, side group replacements should be performed so as tomaintain the torsion angle between Cα and Cβ. Energy minimization may beaccomplished by the AMBER forcefield (Weiner et al, J. Amer. Chem. Soc.106: 765 (1984) using the convergent method. Potentially critical FR-CDRinteractions can be determined by initially modeling the light and heavyvariable chains of mLL2. All FR residues within a 4.5 Å radius of allatoms within CDRs can thereby be identified and retained in the finaldesign model of hLL2.

[0049] The homologous molecular model of Fab fragment of hLL2 wascreated with QUANTA protein modeling package using the x-ray structureof humanized anti-p185her2 antibody fragments (1FVD) as main template.See Carter et al., Proc. Natl. Acad. Sci. 89: 4285 (1992); Eizenbrot etal., J. Mol. Biol. 229: 969 (1993). The sequence identity between thetwo antibodies is about 80%. The insertion regions were modeled bysearching available protein data libraries. After all coordinates weregenerated and connection regions were regularized, a series of energyminimizations were applied to the model. This includes 100 step Steepestdescent (SD) and Conjugated Gradient (CG) EM for side chain atoms only,then 100 step SD and CG EM for all except Cα atoms and finally 100 stepSD and EM for all atoms. A distance related dielectric constant, 4r (ris the atom-atom distance in A) was used for electrostatic interactions.The RMS of atomic position for equivalent main chain and side chainatoms between 1FVD and hLL2 were 1.46 Å and 2.11 Å, respectively. Pointmutations were then applied to hLL2 to generate the models of mutantantibodies, hLL2HCN1 and hLL2HCN5. Complex-type oligosaccharides weremodeled using the same program with the compositions and structureselucidated from carbohydrate sequencing.

[0050] Each generated oligosaccharide chain was then anchored to thecorresponding N-linked glycosylation site with the 01 of the terminalGlcNac superimposed to the N_(d) of the Asn and 01C1 bond of the GlcNacco-lined with one of Nd-H bonds of the Asn. The conformation of theattached oligosaccharide chain was sequentially manipulated so that thelongest branch was close to the variable region of the heavy chain ofhLL2. After each adjustment, 100 step SD and CG EM were applied to sugaratoms with fixed anchor atoms and hLL2 atoms.

[0051] The designs for the CK and CH1 glycosylation sites are based onthe following principles:

[0052] 1. A carbohydrate-addition-site with the sequence NXS/T waschosen. X can be any amino acids except Proline and Aspartate. Wheneverpossible, only single amino acid changes to install potentialglycosylation sites at a chosen position were attempted so as tominimize perturbation of the domain structure.

[0053] 2. Potential CK or CH1-associated glycosylation sites can beidentified from known antibodies sequence of different species orisotypes.

[0054] 3. Analyses of the known structures of human CK and CH1 domainsby computer modeling to identify exposed positions where potentialAsn-glycosylation sites can be planted.

[0055] Based on computer modeling studies, the closest approach distancebetween the VK-appended oligosaccharide and the CDRs was estimated to be20 Å. A distance greater than 4.1 Å is considered to be free ofinteractions. Thus, glycosylation sites which are 4.1 Å or further awayfrom the antigen binding site are likely candidates for use asconjugation sites for antibody fragments. Whenever possible, themutations introduced into the CH1 and CK domains are conservative innature, so as to maintain the final tertiary structure of the proteindomains. A conservative mutation generally involves substitution of onefor another by similar size and clinical properties. Specifically, thedesired sequence is NXT/S. For example, replacement of a glutamine (Q)in the original sequence with asparagine (N) would be considered aconservative substitution. In this way, various CH1 and CK domainmutations can be designed to produce inventive glycosylation sites.

[0056] Only exposed sites will have the chance of being glycosylated.Therefore, computer modeling to help locating additional sites that areat potentially favorable positions was employed. The glycosylation siteHCN5 was predicted to be farther away from the ABS and at the surfaceposition; HCN5 site is located at the bottom loop formed between the Eand F-stands. Other sites, which are “evenly” dispersed along the Cκ andCH1, domains sequences, were randomly selected. In all cases, possibleperturbations in the final tertiary structure were minimized bycarefully choosing sequences that required only one single amino acidsubstitution to become potential glycosylation site. A total of fiveCH₁, (HCN1-5) and four Cκ (KCN1-4)-appended sites were introduced to theCH₁, and Cκ domains, respectively. None of these sites appeared to be“buried,” or at the interface between two juxtaposed domains, asconfirmed by computer modeling analyses.

[0057] N-glycosylation was described only as an example. The principlesinvolved are equally applicable to O-glycosylation. An artisan skilledin the art would readily understand the application of the modeling, thedesign of glycosylation sites, and alteration of constant K, CH, and VKregions, to allow for O-glycosylation. O-glycosylation is known to occurat either threoine or serine. The acceptor sequence for O-linkedglycosylation is relatively ill defined (Wilson et al., Biochem. J. 275:526 (1991). There could be a bias for higher content of proline, serineand threonine in these regions, but accessibility, rather than the exactprimary sequence determines whether a particular threonine or serineresidue will be O-glycosylated. Nevertheless, potential O-glycosylationsequences, such as those identified in other antibodies known to haveO-glycosylation (Chandrashekarkan et al., J. Biol. Chem. 259: 1549(1981); Smyth and Utsumi, Nature 216: 322 (1967); Kim et al., J. Biol.Chem. 269: 12345 (1994), can be used as the standard sequences forgrafting into different positions in the antibodies of interest. Thoseconfirmed to contain extensive O-glycosylation can then be tested asconjugation site.

[0058] Another important aspect of the invention is that once aglycosylation site is identified, further identification of otherpotential glycosylation sites is made easier. This is due to twophenomena. For one, successful glycosylation confirms and helps furtherrefine the modeling of the relevant regions. Secondly, the constant Kand CH1 regions are understood to display considerable symmetry.Therefore, identification of a site where glycosylation occurs on, sayCH1, leads to an expectation that the equivalent CK, position would be agood glycosylation site.

[0059] Light chain mutations. Potential N-linked glycosylation sequenceshave been identified in the kappa constant regions of rabbit antibodiesat aa position 161-163 and 174-176. Similar sites can be introduced intothe CK domain of hLL2. See FIG. 12 for examples.

[0060] Heavy chain mutations. In CH1, a carbohydrate-addition-sequence,Asn-Asn-Ser, has been identified at a.a. positions 161-163 (Kabat'snumbering; Kabat et al., 1991) in some of the human IgM CH1 domains.Similarly, the sequence Asn-Val-Thr, was positioned in a.a. positions168-170 in the CH1 domain of human IgA. Examples of sequences which canbe modified to produce altered glycosylation sites are: mutating thehuman IgG1 sequence Asn-Ser-Gly to Asn-Ser-Val at a.a. positions162-164, Ala-Leu-Thr to Asn-Leu-Thr at a.a. positions 165-167, andLeu-Thr-Ser to Asn-Thr-Ser at a.a. positions 166-168, respectively.These three potential N-linked glycosylation sites, are analogous tothat of IgM and IgA and can be introduced into the CH1 domain of humanIgG1, with expectation of minimal interference on the resultantstructure. Such glycosylation sites may thus remain in a “natural”position. The design of similar mutations is well within one of skill inthe art, based on the teachings in the specification.

[0061] Site-Directed Mutagenesis

[0062] Detailed protocols for oligonucleotide-directed mutagenesis andrelated techniques for mutagenesis of cloned DNA are well-known. Forexample, see Sambrook et al., supra, and Ausubel et al., supra.

[0063] Asn-linked glycosylation sites may be introduced into antibodiesusing conventional site-directed oligonucleotide mutagenesis reactions.For example, to introduce an Asn in position 18 of a kappa protein, onemay alter codon 18 from AGG to AAC. To accomplish this, a singlestranded DNA template containing the antibody light chain sequence isprepared from a suitable strain of E. coli (e.g., dut⁻ ung⁻) in order toobtain a DNA molecule containing a small number of uracils in place ofthymidine. Such a DNA template can be obtained by M13 cloning or by invitro transcription using a SP6 promoter. See, for example, Ausubel etal., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,NY, 1987. An oligonucleotide complementary to the single stranded DNA,comprising the mutated sequence is synthesized conventionally, annealedto the single-stranded template and the product treated with T4 DNApolymerase and T4 DNA ligase to produce a double-stranded DNA molecule.Transformation of wild type E. coli (dut⁺ ung⁺) cells with thedouble-stranded DNA allows recovery of mutated DNA.

[0064] Alternatively, an Asn-linked glycosylation site can be introducedinto an antibody light chain using an oligonucleotide containing thedesired mutation, any amplifying of the oligonucleotide by PCR, andcloning it into the variable regions for the VL chain, or by using RNAfrom cells that produce the antibody of interest as a template. Alsosee, Huse, in ANTIBODY ENGINEERING: A PRACTICAL GUIDE, Boerrebaeck, ed.,W.H. Freeman & Co., pp 103-120, 1992. Site-directed mutagenesis can beperformed, for example, using the TRANSFORMER™ kit (Clontech, Palo Alto,Calif.) according to the manufacturer's instructions.

[0065] Alternatively, a glycosylation site can be introduced bysynthesizing an antibody chain with mutually priming oligonucleotides,one such containing the desired mutation. See, for example, Uhlmann,Gene 71: 29 (1988); Wosnick et al., Gene 60: 115 (1988); Ausubel et al.,above, which are incorporated by reference.

[0066] Although the description above referred to the introduction of anAsn glycosylation site in position 18 of the light chain of an antibody,it will occur to the skilled artisan that it is possible to introduceAsn-linked glycosylation sites elsewhere in the light chain or in theheavy chain variable region, or in the constant regions.

[0067] The presence of a glycosylation site, or the absence of such sitein a humanized Ab, where the site was glycosylated in the murinecounterpart, may or may not affect the binding affinity or specificityof the antibody. Glycosylation sites therefore can be introduced orremoved, by methods described above, but their impact on activity needsto be determined. For reasons discussed above, engineering glycosylationsites in the CH1 or CK regions are preferred.

[0068] General Techniques for RNA Isolation, cDNA Synthesis andAmplification

[0069] RNA isolation, cDNA synthesis, and amplification can be carriedout as follows. Total cell RNA can be prepared from a LL2 hybridoma cellline, using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL., Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random primers can beannealed to 5 μg of RNA in the presence of 2 μl of 10× synthesis buffer[200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA], 1 μlof 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScriptreverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 min followed by incubation at 42° C.for 50 min. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 min.

[0070] Constructing antibodies with engineered glycosylation sites inthe VL and VH regions

[0071] cDNAs encoding the VL and VH regions of the mLL2 mAb have beenisolated and recombinantly subcloned into mammalian expression vectorscontaining the genes encoding kappa and IgG₁ constant regions,respectively, of human antibodies. Cotransfection of mammalian cellswith these two recombinant DNAs expressed a cLL2 mAb that, like theparent mLL2 mAb, bound avidly to, and was rapidly internalized byB-lymphoma cells.

[0072] The CDRs of the VK and VH DNAs have been similarly recombinantlylinked to the framework (FR) sequences of the human VK and VH regions,respectively, which are subsequently linked, respectively, to the humankappa and IgG₁ constant regions, and expressed hLL2 in mammalian cells.

[0073] Once the sequences for the hLL2 VK and VH domains are designed,CDR engrafting can be accomplished by gene synthesis using longsynthetic DNA oligonucleotides as templates and amplifying the longoligonucleotides by PCR, using short oligonucleotides as primers. Inmost cases, the DNA encoding the VK or VH domain will be approximately350 base pairs (bp) long. By taking advantage of codon degeneracy, aunique restriction site may easily be introduced, without changing theencoded amino acids, at regions close to the middle of the V gene DNAsequence. For example, at DNA nucleotide positions 157-162 (amino acidpositions 53 and 54) for the hLL2 VH domain, a unique AvrII site can beintroduced while maintaining the originally designed amino acid sequence(FIG. 4B). Two long non-overlapping single-stranded DNA oligonucleotides(˜150 bp) upstream and downstream of the AvrII site (see, for example,oligo A and oligo Bin in Example 3 below) can be generated by automatedDNA oligonucleotide synthesizer (Cyclone Plus DNA Synthesizer,Milligen-Biosearch). The yields of full length DNA oligonucleotides suchas oligos A and B may be expected to be low. However, they can beamplified by two pairs of flanking oligonucleotides in a PCR reaction.The primers can be designed with the necessary restriction sites tofacilitate subsequent subcloning. Primers for oligo A and for oligo Bshould contain overlapping sequence at the AvrII site so that theresultant PCR product for oligo A and B, respectively, can be joinedin-frame at the AvrII site to form a full length DNA sequence (ca 350bp) encoding the hLL2 VH domain. The ligation of the PCR products foroligo A (restriction-digested with PstI and AvrII) and B(restriction-digested with AvrII and BstEII) at the AvrII site and theirsubcloning into the PstII/BstEII sites of the staging vector, VHpBS, canbe completed in a single three-fragment-ligation step. See for Example3. The subcloning of the correct sequence into VHpBS can be firstanalyzed by restriction digestion analysis and subsequently confirmed bysequencing reaction according to Sanger et al., Proc. Natl. Acad. Sci.USA 74: 5463 (1977).

[0074] The HindIII/BamHI fragment containing the Ig promoter, leadersequence and the hLL2 VH sequence can be excised from the staging vectorand subcloned to the corresponding sites in a pSVgpt-based vector, pG1g,which contains the genomic sequence of the human IgG constant region, anIg enhancer and a gpt selection marker, forming the final expressionvector, hLL2pG1g. Similar strategies can be employed for theconstruction of the hLL2 VK sequence. The restriction site chosen forthe ligation of the PCR products for the long oligonucleotides (oligos Cand D, see examples below) can be NruI in this case.

[0075] The DNA sequence containing the Ig promoter, leader sequence andthe hLL2 VK sequence can be excised from the staging vector VKpBR bytreatment with BamH1/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, to form the finalexpression vector, hLL2pKh.

[0076] Humanization sometimes results in a reduction or even loss ofantibody affinity. Therefore, additional modification might be requiredin order to restore the original affinity. See, for example, Tempest etal., Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988), which are incorporated by reference. Knowing that cLL2 exhibitsa binding affinity comparable to that of its murine counterpart (seeExample 5 below), defective designs, if any, in the original version ofhLL2 can be identified by mixing and matching the light and heavy chainsof cLL2 to those of the humanized version. SDS-PAGE analysis of thedifferent mix-and-match humanized chimeric LL2 under non-reducing (thedisulfide L-H chain connections remain intact) and reducing conditions(the chains separate) permits analyses of the relationships of thedifferent types of light and heavy chains on the properties of themolecule. For example, migration as multiple bands or as a higherapparent molecular size can be due to the presence of a glycan group atthe N-linked glycosylation site found in the FR1 region of the murine VKdomain of LL2. A discrete band migrating at about 25 kDa is the expectedmolecular size for a non-glycosylated light chain.

[0077] In general, to prepare cLL2 mAb, VH and VK chains of mLL2 can beobtained by PCR cloning using DNA products and primers. Orlandi et al.,infra, and Leung et al., infra. The VK PCR primers may be subcloned intoa pBR327-based staging vector (VKpBR) as described above. The VH PCRproducts may be subcloned into a similar pBluescript-based stagingvector (VHpBS) as described above. The fragments containing the VK andVH sequences, along with the promoter and signal peptide sequences, canbe excised from the staging vectors using HindIII and BamHI restrictionendonucleases. The VK fragments which are about 600 bp can be subclonedinto a mammalian expression vector, pKh for example, by conventionalmethods. pKh is a pSVhyg-based expression vector containing the genomicsequence of the human kappa constant region, an Ig enhancer, a kappaenhancer and the hygromycin-resistant gene. Similarly, the about 800 bpVH fragments can be subcloned into pG1g, a pSVgpt-based expressionvector carrying the genomic sequence of the human IgG1 constant region,an Ig enhancer and the xanthine-guanine phosphoribosyl transferase (gpt)gene. The two plasmids may be transfected into mammalian expressioncells, such as Sp2/0-Ag14 cells, by electroporation and selected forhygromycin resistance. Colonies surviving selection are expanded, andsupernatant fluids monitored for production of cLL2 mAb by an ELISAmethod. A transfection efficiency of about 1-10×10⁶ cells is desirable.An antibody expression level of between 0.10 and 2.5 μg/ml can beexpected with this system.

[0078] General Techniques for RNA Isolation, cDNA Synthesis andAmplification

[0079] RNA isolation, cDNA synthesis, and amplification can be carriedout as follows. Total cell RNA can be prepared from a LL2 hybridoma cellline, using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL., Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random primers can beannealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesis buffer[200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA], 1 μlof 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScriptreverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 min followed by incubation at 42° C.for 50 min. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 min.

[0080] Amplification of VH and VK sequences. The VK and VH sequences forcLL2 or hLL2 can amplified by PCR as described by Orlandi et al., (Proc.Natl. Acad. Sci., USA, 86: 3833 (1989)) which is incorporated byreference. VK sequences may be amplified using the primers CK3BH andVK5-3 (Leung et al., BioTechniques, 15: 286 (1993), which isincorporated by reference), while VH sequences can be amplified usingthe primer CH1B which anneals to the CH1 region of murine 1gG, andVHIBACK (Orlandi et al., 1989 above). The PCR reaction mixturescontaining 10 μl of the first strand cDNA product, 9 μl of 10× PCRbuffer [500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM MgCl2, and 0.01%(w/v) gelatin] (Perkin Elmer Cetus, Norwalk, Conn.), can be subjected to30 cycles of PCR. Each PCR cycle preferably consists of denaturation at94° C. for 1 min, annealing at 50° C. for 1.5 min, and polymerization at72° C. for 1.5 min. Amplified VK and VH fragments can be purified on 2%agarose (BiORad, Richmond, Calif.). See Example 3 for a method for thesynthesis of an oligo A (149-mer) and an oligo B (140-mer) on anautomated Cyclone Plus DNA synthesizer (Milligan-Biosearch).

[0081] PCR products for VK can be subcloned into a staging vector, suchas a pBR327-based staging vector VKpBR that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the VK PCR products. PCR products for VH can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al., Proc. Natl.Acad. Sci., USA, 74: 5463 (1977) which, is incorporated by reference.

[0082] Furthermore, it was found that the presence of glycosylationsites, and therefore of appended carbohydrate (CHO) moieties causesefficient and superior conjugation of drugs and chelates. This isespecially true when antibody fragments devoid of CH2-appended CHO arebeing utilized.

[0083] The DNA sequences described herein include all alleles, mutantsand variants thereof, whether occurring naturally or experimentallycreated.

[0084] Production of Antibodies with Mutated CH1 and CK Regions

[0085] CH1 and CK DNA sequences can be isolated, the protein sequencemodeled, and the DNA mutated by methodologies similar to these describedfor the VK and VH sequences. Once the CH1 or CK nucleotide sequence hasbeen excised from a light or heavy chain clone, and a glycosylation siteinserted via mutagenesis, the mutated CH1 or CK sequence can bere-inserted into the corresponding heavy or light chain vector. In thecase of a CH1 mutant, it can be coexpressed with a kappa chainexpression vector, such as hLL2pKh, into an appropriate cell, e.g.,myeloma Sp2/0-Ag14, and colonies can be selected for hygromycinresistance. The supernatant fluids can be monitored for production ofcLL2, hLL2, or LL2 engineered with glycosylation sites in the non Fcconstant regions according to the invention by, for example, an ELISAassay, as described below.

[0086] Transfection, and assay for antibody secreting clones by ELISA,can be carried out as follows. About 10 μg of hLL2pKh (light chainexpression vector) and 20 μg of hLL2pG1g (heavy chain expression vector)can be used for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BiORad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

[0087] The level of expression of an Ig gene containing clone could beenhanced by amplifying the copy number. This is typically done byselection for a selectable marker linked to the gene of interest, herethe Ig gene. One skilled in the art would be familiar with the use ofsuch selection. Often the selective marker is the dihydrofolatereductase gene (dhfr). Typically, a clone that appears to contain anamplified copy number of the gene is identified by its expression andamplification is confirmed by nucleic acid hybridization experiments.Multiple rounds of selection assay and confirmation by hybridization aretypically undertaken.

[0088] Transfectoma clones that are positive for the secretion of cLL2,hLL2, or LL2 engineered with glycosylation sites in the non Fc constantregions according to the invention can be identified by ELISA assay.Briefly, supernatant samples (100 μl) from transfectoma cultures areadded in triplicate to ELISA microtiter plates precoated with goatanti-human (GAH)-IgG, F(ab′)₂ fragment-specific antibody (JacksonImmunoResearch, West Grove, Pa.). Plates are incubated for 1 h at roomtemperature. Unbound proteins are removed by washing three times withwash buffer (PBS containing 0.05% polysorbate 20). Horseradishperoxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.) are added to the wells, (100μl of antibody stock diluted×10⁴, supplemented with the unconjugatedantibody to a final concentration of 1.0 μg/ml). Following an incubationof 1 h, the plates are washed, typically three times. A reactionsolution, [100 μl, containing 167 μg of orthophenylene-diamine (OPD)(Sigma, St. Louis, Mo.), 0.025% hydrogen peroxide in PBS], is added tothe wells. Color is allowed to develop in the dark for 30 minutes. Thereaction is stopped by the addition of 50 μl of 4 N HCl solution intoeach well before measuring absorbance at 490 nm in an automated ELISAreader (Bio-Tek instruments, Winooski, Vt.). Bound antibodies are thandetermined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburgh, Scotland).

[0089] Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbencies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

[0090] Comparative binding affinities of the antibodies thus isolatedmay be determined by direct radioimmunoassay. An cLL2, hLL2, or LL2engineered with glycosylation sites in the non Fc constant regionsaccording to the invention can be used. Antibodies can be labeled with¹³¹I, or ¹²⁵I, using the chloramine T method (see, for example,Greenwood et al., Biochem. J, 89: 123 (1963) which is incorporated byreference). The specific activity of the iodinated antibody is typicallyadjusted to about 10 μCi/μg. Unlabeled and labeled antibodies arediluted to the appropriate concentrations using reaction medium (HSFMsupplemented with 1% horse serum and 100 μg/ml gentamicin). Theappropriate concentrations of both labeled and unlabeled antibodies areadded together to the reaction tubes in a total volume of 100 μl. Aculture of Raji cells is sampled and the cell concentration determined.The culture is centrifuged and the collected cells washed once inreaction medium followed by resuspension in reaction medium to a finalconcentration of about 10⁷ cells/ml. All procedures are carried out inthe cold at 4° C. The cell suspension, 100 μl, is added to the reactiontubes. The reaction is carried out at 4° C. for 2 h with periodic gentleshaking of the reaction tubes to resuspend the cells. Following thereaction period, 5 ml of wash buffer (PBS containing 1% BSA) is added toeach tube. The suspension is centrifuged and the cell pellet washed asecond time with another 5 ml of wash buffer. Following centrifugation,the amount of remaining radioactivity remaining in the cell pellet isdetermined in a gamma counter (Minaxi, Packard Instruments, Sterling,Va.).

[0091] The antigen-binding property of the antibodies of the inventioncan be evaluated by competition binding with labeled mLL2 for an LL2anti-idiotype antibody (WN).

[0092] The Raji cell surface antigen binding affinities of mix-and-matchand fully humanized antibodies can be compared to that of cLL2 usingvarious concentrations of mLL2 F(ab′)₂ fragments devoid of the Fcportion as competitors, as evaluated by flow cytometry assay. Residualsurface-bound LL2 antibodies carrying the human Fc portions (cLL2 andmix-and-match LL2) can be detected by a FITC-labeled anti-human Fcspecific antibody in a flow cytometry assay. Where mix-and-match LL2antibodies exhibit antigen-binding affinities similar to that of cLL2,it can be concluded that the original designs for the humanization ofboth the light and heavy chains retain the mLL2 immunoreactivity.

[0093] The internalization of cLL2, hLL2, or LL2 engineered withglycosylation sites in the non Fc constant regions according to theinvention into target cells can be followed by fluorescence labeling,essentially according to the procedure of Pirker et al., J. Clin.Invest., 76: 1261 (1985), which is incorporated by reference. CulturedRaji cells are centrifuged and the cells resuspended in fresh medium toa concentration of about 5×10⁶ cells/ml. To each well of a 96-wellmicrotiter plate, 100 μl of the cell suspension is added. Theantibodies, 40 μg/ml, in a volume of 100 μl are added to the reactionwells at timed intervals so as to terminate all reactionssimultaneously. The plate is incubated at 37° C. in a CO₂ cell cultureincubator. Unbound antibodies are removed by washing the cells threetimes with cold 1% FCS/PBS at the end of the incubation. The cells arethen treated with 1 ml of Formaid-Fresh [10% formalin solution (Fisher,Fair Lawn, N.J.)] for 15 min at 4° C. After washing, antibodies presenteither on the cell surface or inside the cells are detected by treatmentwith FITC-labeled goat anti-mouse antibody (Tago, Burlingame, Calif.),or FITC-labeled goat anti-human antibody (Jackson ImmunoResearch, WestGrove, Pa.), depending on whether the antibody being assayed for ismurine, chimeric, or humanized, respectively. Fluorescence distributionsare evaluated using a BH-2 fluorescence microscope (Olympus, LakeSuccess, N.Y.).

[0094] The rate of antibody internalization can be determined accordingto Opresko et al., (J. Biol. Chem., 262: 4116 (1987)), usingradio-iodinated antibody as tracer. Briefly, radiolabelled antibodies(1×10⁴ cpm) are incubated with the Raji cells (1×10⁶ cells/ml) at 4° C.for 2 h in 0.5 ml of DMEM medium containing 1% human serum. Followingthe reaction interval, non-specifically bound antibodies are removed bywashing three times with 0.5 ml of DMEM medium. To each of the reactiontubes 0.5 ml of DMEM medium is added and the suspension incubated at 37°C. for the determination of internalization. At timed intervals,triplicates of cells are removed and chilled immediately in an ice bathto stop further internalization. Cells are centrifuged at 1000×g for 5min at 4° C. The supernatant is removed and counted for radioactivity.The surface-bound radioactivity is removed by treatment with 1 ml 0.1 Macetate/0.1 M glycine buffer at pH 3.0 for 8 min. in the cold.Radioactivity removed by the acid treatment, and that remainingassociated with the cells, are determined. The ratio of theCPM_(internalization)/CPM_(surface) is plotted versus time to determinethe rate of internalization from the slope.

[0095] The representative embodiments described below are simply used toillustrate the invention. Those skilled in these arts will recognizethat variations of the present materials fall within the broad genericscope of the claimed invention. The contents of all references mentionedherein are incorporated by reference.

EXAMPLE 1

[0096] Choice of Human Frameworks and Sequence Design for theHumanization of LL2 Monoclonal Antibody

[0097] By comparing the murine variable (V) region framework (FR)sequences of LL2 to that of human antibodies in the Kabat data base(Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed.,U.S. Department of Health and Human Services, U.S. Government PrintingOffice, Washington, D.C.), which is incorporated by reference, the humanREI (FIG. 1A,) and EU (FIG. 1B) sequences were found to exhibit thehighest degree of sequence homology to the FRs of VK and VH domains ofLL2, respectively. Therefore, the REI and EU FRs were selected as thehuman frameworks onto which the CDRs for LL2 VK and VH were grafted,respectively. The FR4 sequence of NEWM, however, rather than that of EU,was used to replace the EU FR4 sequence for the humanization of LL2heavy chain. Based on the results of computer modeling studies (FIGS. 2Aand 2B), murine FR residues having potential CDR contacts, which mightaffect the affinity and specificity of the resultant antibody, wereretained in the design of the humanized FR sequences (FIG. 1).

[0098] Two versions of humanized heavy chain were constructed. In thefirst version (hLL2-1), the glutamine (Q) at amino acid position 5(Kabat numbering) was introduced to include a PstI restriction site tofacilitate its subcloning into the staging vector (FIG. 3). This murineresidue was converted, by oligo-directed mutagenesis, to the human EUresidue valine (V) in hLL2-2. It should be noted that in the originalmurine kappa chain variable sequence, a potential N-linked glycosylationsite was identified at positions 18-20 and was used for carbohydrateaddition. This glycosylation site was not included in the REI FRsequence used for LL2 light chain humanization.

EXAMPLE 2

[0099] PCR Cloning and Sequence Elucidation for LL2 Heavy and LightChain Variable Regions

[0100] The variable regions for both heavy (VH) and light (VK) chains ofmLL2 (IgG2a) were obtained by PCR cloning using DNA primers as describedin general above and in greater detail in Example 3, below. As PCR isprone to mutations, the variable region sequence of multiple individualclones for either the heavy or light chains was determined for sixclones and confirmed to be identical prior to use for the constructionof the chimeric antibody.

[0101] The PCR products for VK were subcloned into a pBR327-basedstaging vector, VKpBR, which contained an Ig promoter, a signal peptidesequence and convenient restriction sites to facilitate in-frameligation of the VK PCR products (FIG. 3A). The PCR products for VH weresubcloned into a similar pBluescript-based staging vector, VHpBS (FIG.3B).

[0102] As noted above, at least six individual clones containing therespective PCR products were sequenced according to the method of Sangeret al., 1977, above. All were shown to bear identical sequences andtheir respective sequences were elucidated, as shown in FIG. 4A for LL2VK and in FIG. 4B for LL2 VH. No defective mutations were identifiedwithin the sequences encoding the VK and VH regions. Comparison of thePCR-amplified variable region sequences of LL2 with the Kabat database(Kabat et al., above) suggested that the VK and VH sequences of LL2belong to subgroup 5 and 2B, respectively. Important residues such asCys for intra-domain disulfide linkage were retained at appropriatepositions.

[0103] In the FR1 framework region of VK, an N-linked carbohydrateattachment site, Asn-Val-Thr, was identified at position 18-20 (FIG.4A), suggesting that the VK of LL2 might be glycosylated. As will bedetailed below, SDS-PAGE analysis under reducing conditions demonstratedthat this Asn glycosylation site is indeed utilized for carbohydrateaddition. The presence of the glycosylation site in the variable regiondoes not, however, appear to affect the immunoreactivity of theantibody. A comparison of the immunoreactivity of mLL2 with that of cLL2in a competitive RIA showed that the two antibodies have nearlyidentical activities.

EXAMPLE 3

[0104] PCR/Gene Synthesis of the Humanized V Genes

[0105] The designed sequence for the hLL2 VH domain, the construction ofthe hLL2 VH domain by long oligonucleotides and PCR, and the stagingvector VHpBS containing the hLL2 VH domain are summarized in the sketchshown in FIG. 6.

[0106] For the construction of the hLL2 VH domain, oligo A (149-mer) andoligo B (140-mer) were synthesized on an automated CYCLONE PLUS DNAsynthesizer (Milligen Bioresearch).

[0107] Oligo A represents the minus strand of the hLL2 VH domaincomplementary to nucleotides 24 to 172 (SEQ ID NO: 22): 5′-TAT AAT CATTCC TAG GAT TAA TGT ATC CAA TCC ATT CCA GAC CCT GTC CAG GTG CCT GCC TGACCC AGT GCA GCC AGT AGC TAG TAA AGG TGT AGC CAG AAG CCT TGC AGG AGA CCTTCA CTG ATG ACC CAG GTT TCT TGA CTT CAG CC-3′.

[0108] Oligo B represents the minus strand of the hLL2 VH domaincomplementary to nt 181 to 320 (SEQ ID NO: 23): 5′-CCC CAG TAG AAC GTAGTA ATA TCC GCA CAA AAA TAA AAT GCC GTG TCC TCA GAC CTC AGG CTG CTC AGCTCC ATG TAG GCT GTA TTG GTG GAT TCG TCT GCA GTT ATT GTG GCC TTG TCC TTGAAG TTC TGA TT-3′

[0109] Oligos A and B were cleaved from the support and deprotected bytreatment with concentrated ammonium hydroxide. After the samples werevacuum-dried (SpeedVac, Savant, Farmingdale, N.Y.) and resuspended in100 μl of water, incomplete oligomers (less than 100-mer) were removedby centrifugation through a CHROMOSPIN-100™ column (Clonetech, PaloAlto, Calif.) before the DNA oligomers were amplified by PCR. Allflanking primers for the separate amplifications and PCR cloning ofoligos A and B were purified by SDS-PAGE essentially according to themethods of Sambrook et al., 1989, above. From the CHROMASPIN-purifiedoligo A, 1 μl of sample stock was PCR-amplified in a reaction volume of100 μl by adding 5 μl of 5 μM of oligo (SEQ ID NO: 24): 5′-CCA GCT GGTCCA ATC AGG GGC TGA AGT CAA GAA ACC TG-3′ and of oligo (SEQ ID NO: 25):5′-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA ATG-3′ in the presence of 10μl of 10×PCR Buffer (500 mM KCl, 100 mM Tris-HCL buffer, pH 8.3, 15 mMMgCl₂) and 5 units of AMPLITAQ™ DNA polymerase (Perkin Elmer Cetus,Norwalk, Conn.). This reaction mixture was subjected to 30 cycles of PCRreaction consisting of denaturation at 94° C. for 1 minute, annealing at50° C. for 1.5 minutes, and polymerization at 72° C. for 1.5 minutes.

[0110] Oligo B was PCR-amplified by the primer pairs: 5′-TAA TCC TAG GAATGA TTA TAC TGA GTA CAA TCA GAA CTT CAA GGA CAA G-3′ (SEQ ID NO: 26)and: 5′-GGA GAC GGT GAC CGT GGT GCC TTG GCC CCA GTA GAA CGT AGT AA-3′(SEQ ID NO: 27) under similar conditions.

[0111] Double-stranded PCR-amplified products for oligos A and B weregel-purified, restriction-digested with PstI/AvrII (PCR product of oligoA) and BstEII/AvrII (PCR product of oligo B), and subcloned into thecomplementary PstI/BstEII sites of the heavy chain staging vector,VHpBS. The humanized VH sequence was subcloned into the pG1g vector,resulting in the final human IgG1 heavy chain expression vector,hLL2pG1g.

[0112] For constructing the full length DNA of the humanized VKsequence, oligo E (150-mer) and oligo F (121-mer) were synthesized asdescribed above. Oligo E comprises (SEQ ID NO: 28): 5′-CCT AGT GGA TGCCCA GTA GAT CAG CAG TTT AGG TGC TTT CCC TGG TTT CTG CTG GTA CCA GGC CAAGTA GTT CTT GTG ATT TGC ACT GTA TAA AAC ACT TTG ACT GGA CTT ACA GCT CATAGT GAC CCT ATC TCC AAC AGA TGC GCT CAG-3′. It represents the minusstrand of the humanized VK domain complementary to nt 31 to 180, andthis sequence was PCR-amplified by oligo (SEQ ID NO: 29): 5′-GAC AAG CTTCAG CTG ACC CAG TCT CCA TCA TCT CTG AGC GCA TCT GTT GGA G-3′ and oligo(SEQ ID NO: 30): 5′-AGA GAA TCG CGA AGG GAC ACC AGA TTC CCT AGT GGA TGCCCA GTA-3′.

[0113] The Oligo F sequence (SEQ ID NO: 31) is 5′-GCA CCT TGG TCC CTCCAC CGA ACG TCC ACG AGG AGA GGT ATT GGT GAC AAT AAT ATG TTG CAA TGT CTTCTG GTT GAA GAG AGC TGA TGG TGA AAG TAA AAT CTG TCC CAG ATC CGC TGCC-3′. It represents the minus strand of the humanized LL2 VK domaincomplementary to nt 208 to 328. It was PCR amplified by oligo (SEQ IDNO:32): 5′-GAC AAG CTT TCG CGA TTC TCT GGC AGC GGA TCT GGG ACA G-3′ andoligo (SEQ ID NO: 33): 5′-GAC CGG CAG ATC TGC ACC TTG GTC CCT CCACCG-3′.

[0114] Gel-purified PCR products for oligos E and F wererestriction-digested with PvuII/NruI and NruI/BglIII, respectively. Thetwo PCR fragments E and F were then joined at the NruI site and ligatedto the complementary PvuI/BcII sites of the light chain staging vector,VKpBR. The humanized VK sequence was subcloned into vector pKh to formthe final human kappa chain expression vector, hLL2pKh.

[0115] To express the humanized antibodies, about 10 μg of linearizedhLL2pKh and 20 μg of linearized hLL2pG1g were used to transfect 5×10⁶SP2/0 cells by electroporation. The transfectomas were selected withhygromycin at 500 μg/ml and secreted antibody was purified on a 1×3 cmcolumn of protein A. After concentrating the purified antibody byCentricon 30 centrifugation, antibody concentration was determined byELISA. The final concentration of the antibody was adjusted to 1 mg/mlin PBS buffer containing 0.01% (w/v) sodium azide as a preservative.

[0116]FIG. 1 compares the amino acid sequence between murine andhumanized LL2 VK domains (FIG. 1A, SEQ ID NOS: 2, 6 & 20) and betweenmurine and humanized LL2 VH domains (FIG. 1B, SEQ ID NOS: 4, 21 & 8). Inthe VK chain, human REI framework sequences were used for all FRs. Inthe VH chain, human EU framework sequences were used for FR 1-3, andNEWM sequences were used for FR-4. Only human FR sequences that aredifferent from that of the mouse are shown. Asterisks indicate murine FRsequences that are different from that of the human FR at correspondingpositions. Murine residues at these positions were retained in thehumanized structure. CDRs are boxed.

[0117] In FIG. 4A (SEQ ID NOS: 1 & 2) there are shown the doublestranded DNA and corresponding amino acid sequences (shown by singleletter code) of the murine LL2 VK domain. CDR 1-3 amino acid sequencesare boxed. The corresponding display for VH is shown in FIG. 4B (SEQ IDNOS: 3 & 4).

[0118] In FIG. 5A (SEQ ID NOS: 5 & 6) and FIG. 5B (SEQ ID NOS: 7 & 8)there are shown double-stranded DNA sequences and amino acid sequencesof humanized LL2 VK and LL2 VH, respectively. Amino acid sequences areshown by the single-letter code, and CDR amino acid sequences are boxed.

EXAMPLE 4

[0119] Construction, Expression and Purification of Chimeric LL2Antibodies

[0120] The fragments containing the VK and VH sequences of LL2, togetherwith the promoter and signal peptide sequences, were excised fromLL2VKpBR and LL2VHpBS, respectively, by double restriction digestionwith HindIII and BamHI. The about 600 bp VK fragments were thensubcloned into the HindIII/BamHI site of a mammalian expression vector,pKh (FIG. 3A). pKh is a pSVhyg-based expression vector containing thegenomic sequence of the human kappa constant region, an Ig enhancer, akappa enhancer and the hygromycin-resistant gene. Similarly, the ca. 800bp VH fragments were subcloned into the corresponding HindIII/BamHI siteof pG1g (FIG. 3B), a pSVgpt-based expression vector carrying the genomicsequence of the human IgG1 constant region, an Ig enhancer and thexanthine-guanine phosphoribosyltransferase (gpt) gene. The finalexpression vectors are designated as LL2pKh and LL2pG1g, respectively.

[0121] The two plasmids were co-transfected into Sp2/0-Ag14 cells byelectroporation and selected for hygromycin resistance. Supernatant fromcolonies surviving selection were monitored for chimeric antibodysecretion by ELISA assay (see above). The transfection efficiency wasapproximately 1-10×10⁶ cells. The antibody expression level, in aterminal culture, was found to vary in the range between <0.10 and2.5/μg/ml.

[0122] Protein A-purified mLL2 and cLL2 were analyzed by SDS-PAGE underreducing and non-reducing conditions. The light chains of both mLL2 andcLL2 showed a higher than expected apparent molecular weight. As thehuman kappa constant region of cLL2 is known to contain no potentialglycosylation site, it can be inferred that the potential glycosylationsite identified in the FR1 region of LL2 VK domain was utilized.Different versions of hLL2 and cLL2 antibodies were analyzed by SDS-PAGEunder reducing and non-reducing conditions. One hLL2 version was hLL2-1(with seven murine FR residues in the VH domain). Another hLL2 versionwas hLL2-2 with 6 murine FR residues in the VH domain. The humanizedlight chains migrated more rapidly and the bands were more discretebands when compared to the chimeric light chains.

[0123] Mix-and-match, cLL2 and hLL2 antibodies were analyzed bySDS-PAGE, under reducing and non-reducing conditions. The mix-and-matchversions analyzed were the (hL/cH)LL2, the (cL/hH)LL2-1, and the(cL/hH)LL-2. (cL/hH)LL2-1 and (cL/hH)LL-2 contain 7 and 6 murineresidues in the FR regions of the heavy chain, respectively. Themigration observed for the (hL/cH)LL2 suggested that the humanized LL2light chain did not undergo glycosylation.

EXAMPLE 5

[0124] Binding of cLL2 Antibody to Raji Cell Surface Antigens

[0125] A competition cell binding assay was carried out to assess theimmunoreactivity of cLL2 relative to the parent mLL2. Using ¹³¹I-labeledmLL2 (0.025 μg/ml) as a probe, Raji cells were incubated with theantibodies and the relative binding to the cells determined from theamount of cell-bound labeled mLL2 (see above). As shown by thecompetition assays described in FIG. 7, both mLL2 and cLL2 antibodiesexhibited similar binding activities.

[0126] The results were confirmed by a second competition assay based onflow cytometry. Briefly, using Raji cells as before and varying theconcentration of one antibody relative to other, as before, the amountof bound mLL2 or cLL2 was determined with FITC-labeled anti-mouse Fc oranti-human Fc antibodies followed by analysis using flow cytometry.

EXAMPLE 6

[0127] Binding of hLL2 Antibodies to Raji Cells

[0128] In experiments similar to those of Example 5, the antigen bindingaffinities of the three different combinations of mix-and-match orhumanized LL2 were compared with that of cLL2 in the flow cytometryassay.

[0129] Briefly, 1 μg of cLL2, mix-and-match LL2, hLL2-1 or hLL2-2antibodies were incubated with 10⁸ Raji cells in the presence of varyingconcentrations of mLL2 F(ab′)₂ fragments (as competitor) in a finalvolume of 100 μl of PBS buffer supplemented with 1% FCS and 0.01% sodiumazide. The mixture was incubated for 30 minutes at 4° C., and washedthree times with PBS to remove unbound antibodies. By taking advantageof the presence of human Fc portions in the antibodies, the bindinglevels of the antibodies were assessed by adding a 20× dilutedFITC-labeled goat anti-human IgG1, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed threetimes with PBS, and fluorescence intensities measured by a FACSCANfluorescence activated cell sorter (Becton-Dickinson, Bedford, Mass.).The results are shown in FIG. 8A. Using the same methods, cLL2 wascompared to two versions of hLL2 (FIG. 8B).

[0130] The results shown in FIGS. 8A and B demonstrate that theimmunoreactivity of cLL2 is similar or identical to that of humanized ormix-and-match antibodies. Taken together with the comparison of cLL2with mLL2 (FIG. 7), the authenticity of the sequences for chimeric andhumanized VK and VH obtained is established, and the functionality ofcLL2 and hLL2 confirmed.

EXAMPLE 7

[0131] Internalization of mLL2 and cLL2 by Raji Cells

[0132] One of the unique characteristics of the LL2 antibody is itsrapid internalization upon binding to Raji cells (Shih et al., 1994above). Murine LL2 after internalization is likely to be rapidlytransferred to the Golgi apparatus and from there to the lysosome, theorganelle responsible for the degradation of a wide variety ofbiochemicals (Keisari et al., Immunochem., 10: 565 (1973)).

[0133] Rates of antibody internalization were determined according toOpresko et al., 1987 above. The ratio ofCPM_(intracellular)/CPM_(surface) was determined as a function of time.

[0134] Rates of LL2 antibody internalization were determined byincubating radiolabelled LL2 antibody (1×10⁶ cpm) with 0.5×10⁶ Rajicells in 0.5 ml of DMEM buffer containing 1% human serum for 2 hrs. at4° C. Excess human serum was included to saturate Raji cell surface Fcreceptors in order to exclude or minimize non-antigen-specificinternalization mediated through the Fc receptors. Unbound radiolabelledLL2 antibodies were removed from the cells by washing three times with0.5 ml portions of DMEM at 4° C. Cells were then incubated at 37° C.,and, at timed intervals, aliquots of the cell suspension weretransferred to ice in order to stop internalization. The cells in thesealiquots were isolated by centrifugation at 1,000×g for 5 mins. at 4°C., and surface bound radiolabelled LL2 stripped off cells with 1 ml of0.1 M glycine acetate buffer, pH 3, for 8 mins. at 4° C. Radioactivitythus obtained (CPM surface) and radioactivity remaining in the cells(CPM intracellular) were determined. Rates of internalization werecalculated from the slope of the plot of intracellular: surfaceradioactivity ratios as a function of time.

[0135] As shown in FIG. 9, mLL2, cLL2, cLL2Q and hLL2 antibodies wereinternalized at a similar rate (Ke=0.107 (mLL2) to 0.1221 (cLL2Q, NVT toQVT mutation). Those numbers suggested that approximately 50% of thesurface-bound antibody could be internalized in 10 min. The results showthat neither chimerization nor humanization nor deglycosylation bymutagenesis of mLL2 antibodies impair rates of internalization.

[0136] The pattern of internalization for mLL2, cLL2 and hLL2 was alsomonitored by fluorescence microscopy on a time-course basis using aFITC-labeled second antibody probe as described in the specification.Internalization of both antibodies was observed in at the earliest timepoint measurable. At 5 minutes, antibodies were seen both on the cellsurface and internalized in areas immediately adjacent to the membraneas cytoplasmic micro-vesicles. At 15 min. post-incubation, the fine dotsdispersed around the intramembrane began to merge into a group ofgranules, at locations believed to be the Golgi apparatus. As moreantibodies were being internalized after 30 min. of incubation,redistribution of the grouped antibodies to scattered locations,probably the lysosome in which the antibodies were degraded, wasobserved. At 2 hrs post-incubation, most of the antibodies were foundinside the cell. Only strong surface staining was observed when LL2 wasincubated for 20 min on ice. Both mLL2 and cLL2 were internalized with asimilar pattern. The internalization of LL2 was associated specificallywith antigen-antibody binding, as the irrelevant control humanizedantibody demonstrated only dull surface staining.

[0137] The A103 antibody (an IgG2a antibody that binds to the surface ofall human epithelial cells but does not internalize efficiently (Matteset al., Hybridoma, 2: 253 (1983)) showed strong membrane staining at upto 2 h, while the anti-transferrin receptor antibody (SF9) internalizedrapidly, just as did LL2.

EXAMPLE 8

[0138] Role of Glycosylation Site in FR1 Region of LL2 VK Sequence

[0139] Of particular inventive interest is the identification of anAsn-glycosylation site at position 18-20 within the FR1 region of theLL2 NVT light chain sequence (FIG. 4A, SEQ ID NOS: 1 & 2). As shownabove, SDS-PAGE analysis under reducing condition suggests that the Asnglycosylation site is utilized for carbohydrate addition. In thisexample, the influence of the carbohydrate moiety at position 18-20 onthe functional activities of the light chains was examined.

[0140] Murine and chimeric LL2 light chains, treated or untreated withendoglycosidases F, were examined by SDS-PAGE under reducing andnon-reducing conditions. There was no distinction between the antibodytypes as to electrophoretic behavior. In both cases, deglycosylationreduced the rate of migration of the light chain.

[0141] The effect of deglycosylation on the binding affinity to Rajicells of the mLL2 antibody is shown in FIG. 10. Removing carbohydrate byendoglycosidases F did not influence the binding activity.

[0142] A mutation was introduced at position 18 of the light chain sothat the Asn was replaced with Gln to produce LL2Q VK FR1. SDS-PAGEanalyses demonstrated that the NVT to QVT mutation abolishedglycosylation of the antibody. Comparison of the Raji cell bindingaffinity for cLL2 with and without light chain VK glycosylationdemonstrated that the carbohydrate moiety did not influence binding ofthe antibody to these cells.

[0143] It can be concluded that the presence of the carbohydrate site inthe variable region does not affect the immunoreactivity of theantibody. Computer modeling studies suggested that the VK carbohydratemoiety in LL2 is remotely positioned from the CDRs and forms a “cap”over the bottom loops of the FR-associated 13-barrels supporting theCDRs. Humanization without inclusion of the original glycosylation siteresulted in a CDR-grafted LL2 antibody with immunoreactivity comparableto that of its murine counterpart. These characteristics indicate thatthe glycosylation site can be used for conjugating therapeutic ordiagnostic agents to LL2 without compromising the ability of theantibody to bind and internalize in B-lymphoma or leukemia cells.

EXAMPLE 9

[0144] Conjugation of LL2 at its VK Region Carbohydrate-Bearing Site

[0145] The apparent lack of involvement of the variable regioncarbohydrate moiety in the functional activities of mLL2, cLL2 and hLL2mAbs indicates that this moiety could profitably be used as the site ofattachment of cytotoxic or detection agents such as radionuclides ortoxins, and thereby avoid potential interference with the binding of theconjugate to a cell surface.

[0146] Using procedures described in Shih et al., U.S. Pat. No.5,057,313 (which is incorporated by reference) for preparing antibodyconjugates through an oxidized carbohydrate moiety of the antibody and aprimary alkylamine group of a polymeric carrier to which are covalentlyone or more of a variety of drugs, toxins, chelator and detectablelabels, a doxorubicin-dextran-LL2 antibody fragment devoid of appendedglycan was produced containing multiple copies of the drug. Thecarbohydrate moieties of the cLL2 VK FR1 region involved were thosecovalently bound to the Asn glycosylation site.

[0147] In one synthesis, dextran (18-40 kDa) was converted to an aminodextran by oxidation of the dextran by NaIO₄, Schiff base formation withNH₂—CH₂—CHOH—CH₂—NH₂, and reduction with NaBH₄. The amino dextran wasthen condensed with doxorubicin (DOX) in the presence of succinicanhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to produceDOX-aminodextran. The latter was then condensed with an aldehydic groupon LL2 VK FR-1 produced by oxidizing the carbohydrate moiety of theantibody fragment with NaIO₄.

[0148] In one preparation of DOX-LL2, the number of moles of DOXattached to dextran was 14 moles per mole dextran, and the number ofmoles of doxorubicin per mole F(ab′)₂ was 8.9. The immunoreactivity inthe Raji cell binding assay above was about 80% of control values. Thisconjugation system is not limited to the mLL2 antibody. In a comparativestudy, 15-19 moles of DOX were bound per mole of cLL2.

[0149] The conjugation possibilities are not limited to the use of acarrier dextran as in the example above. For example, the carbohydratemoiety of the LL2 VK FR1 region can be oxidized to produce aldehydicgroups. These in turn can be reacted with an amino group on any drug toproduce a Schiff base which, upon reduction, produces multiple copies ofthe drug stably linked to the antibody via alkylamine groups.

[0150] For example, where the drug is aminohexyl DTPA (a chelatingagent), there is produced a LL2 covalently bound to a chelator. Thechelator can be used to deliver to target tissues, for example, aradionuclide or paramagnetic metal ion, with a potential for diagnosticand therapeutic uses. DTPA-LL2 conjugates were produced containing 5.5moles of the chelator/mole of antibody which, in turn, chelated 47.3% ofY-90 and 97.4% In-111

EXAMPLE 10

[0151] Enhanced Production of a Humanized Anti-B-Cell Lymphoma Antibody.

[0152] Despite a demonstrated efficacy for murine LL2 in the treatmentand diagnosis of non-Hodgkin's B-cell lymphoma, a thorough study of theclinical significance of its humanized version (hLL2), however, wasrendered difficult due to the low hLL2 productivity of the originaltransfectoma (ca. 1 mg/liter in a terminal culture). By re-ligating thehLL2 heavy and light chain sequences into an expression vectorcontaining an amplifiable dihydrofolate reductase gene(dhfr)(hLL2pdHL2), we were able to transfect the vector into SP2/0 cellsby electroporation and generate a methotrexate (MTX) resistant and hLL2producing clone. At a MTX concentration of 0.1 μM, 1.4 mg of hLL2 werepurified from a one-liter terminal culture. The level of hLL2 productionrose with stepwise increases in the concentration of MTX in the culturemedia, and reached a production plateau of 70+/−5 mg/liter at 3 μM ofMTX. The hLL2 thus purified exhibited a PI of 10.3 with conservedimmunoreactivity. Furthermore, complete removal of MTX selection, andfreezing and thawing did not appear to affect the high levelproductivity of the established clone, suggesting that the amplifiedgenes were stably integrated into the chromosome.

EXAMPLE 11

[0153] Construction of N-Linked Glycosylation Sites into the ConstantRegion of hLL2 Antibody

[0154] 1. Designing N-linked glycosylation site mutations.

[0155] (1) Light Chain Mutations.

[0156] Potential N-linked glycosylation sequences have been identifiedin the kappa constant regions of rabbit antibodies at a.a. position161-163 and 174-176. Similar sites can be introduced into the CK domainof hLL2, designated as sites KCNI and KCN2, respectively. Additionally,three other CK mutants, namely KCN3 and KCN4 were designed, as listed inFIG. 12.

[0157] (2) Heavy Chain Mutations.

[0158] Human IgM contains potential carbohydrate-addition-sequence, NNS,in the CH1 domain at amino acid position 161-163. Similarly, thesequence, NVT, was positioned at the residues 168-170 in the CH, domainof human IgA. By the same rationale used in the designs of light chainmutations, certain heavy chain mutations also were introduced (FIG. 12).

[0159] Carbohydrate-addition-sequence, Asn-Asn-Ser, was identified ata.a. positions 161-163 (Kabat's numbering; Kabat et al., 1991) in someof the human IgM CH1 domains. Similarly, the sequence, Asn-Val-Thr, waspositioned in a.a. positions 168-170 in the CH1 domain of human IgA. Bymutating the human IgG1 sequence Asn-Ser-Gly to Asn-Ser-Val at a.a.positions 162-164, Ala-Leu-Thr to Asn-Leu-Thr at a.a. positions 165-167,and Leu-Thr-Ser to Asn-Thr-Ser at a.a. positions 166-168, respectively,three potential N-linked glycosylation sites, most analogous to that ofIgM and IgA, were introduced into the CH1 domain of human IgG1, withminimal interference on the resultant structure. Such glycosylationsites may thus remain in a “natural” position. Other glycosylationacceptor sequences were introduced based on their surface accesibilityas predicated by computer modeling (HCM5, for example). Yet other siteswere chosen randomly, by facility to mutate the sequence, withoutmodeling.

[0160] 2. Engineering Mutation Constructs for Expression.

[0161] (1) Design and Synthesis of Primers for Mutagenesis.

[0162] Oligonucleotide-directed site specific mutagenesis was used tointroduce the designed potential N-linked glycosylation sites in hLL2antibody. The oligonucleotide primers corresponding to each CK and CHImutation were synthesized and used for in vitro mutagenesis. Each ofthese primers also introduced into the target DNA fragment a restrictioncleavage site (Table 1, underlined sequences) to facilitate subsequentscreening process. In Table 1, the bold letters indicate the mutatedbases.

[0163] Table 1 CK mutation primers: CKN1 (SEQ ID NO: 34) 5′-CCAATCGGGTAATTCGAA TGAGAGTGTCACAGAG-3′ CKN2 (SEQ ID NO: 35) 5′-GGACAGCACCTACAACTTAAGCAGCACCCTGAC-3′ CKN3 (SEQ ID NO: 36) 5′-GGAAGGTGGATAACGCGTCCCAATCGGGTAA-3′ CKN4 (SEQ ID NO: 37) 5′-AGCAGCACCCTAAATTTGAGCAAAGCAGACT-3′ CKN5 (SEQ ID NO: 38) 5′-GAGTGTCACAGAG AACGTTAGCAAGGACAGCACC-3′ CH₁ mutation primers: HCN1 (SEQ ID NO: 39)5′-GTGTCGTGGAACTCA AGCGCT CTGACCAGCGGC-3′ HCN2 (SEQ ID NO: 40)5′-TTCCCGGCTGTCCT GAATTCCTCAGGACTCTACT-3′ HCN3 (SEQ ID NO: 41)5′-CCTCAGGACTCTACTC GAATTC CAGCGTGGTGACCGT-3′ HCN4 (SEQ ID NO: 42)5′-GTGGTGACCGTCCC GAATTC CAGCTTGGGCACC-3′ HCN5 (SEQ ID NO: 43)5′-GCCCTCCAGCAGCAAC GGTACCCAGACCTACATCTGC-3′

[0164] (2) Construction of Expression Vectors.

[0165] By in vitro site-specific mutagenesis, potential N-linkedglycosylation sequences were introduced into the genes encoding thelight and heavy chain of hLL2. The sequences were confirmed by DNAsequencing. Each mutated gene was then subcloned into the correspondingexpression vector (hLL2pKh for the kappa chain and hLL2pG1g for theheavy chain).

[0166] The CH1 domain of human IgG1 was first excised from theexpression vector LL2pG1g containing the human genomic IgG1 constantregion sequence (Leung et al., 1994b) by digestion with the restrictionenzymes BamHI and BstXI, and subcloned into the corresponding sites ofthe pBluescript SK vector (Stratagene, La Jolla, Calif.) for furthermanipulations. The resultant vector is designated as CH1pBS.

[0167] Mutations were accomplished using the Transformer™ Site-DirectedMutagenesis Kit (CLONTECH, Palo Alto, Calif.) according to themanufacturer□s specifications. The selection primer, MutKS (5′-ACG GTATCG ATA TGC ATG ATA TCG AAT T-3′), is designed for use in conjunctionwith the respective mutation primers in all cases. It was chosen toconvert the HindIII restriction site in the cloning sequence ofpBluescript to a NsiI restriction site (underlined).

[0168] To mutate Asn-Ser-Gly to Asn-Ser-Thr at a.a. positions 162-164,the selection primer MutKS and the primer CHO162 (5′-GTG TCG TGG AAT TCAACC GCC CTG ACC AGC GGC-3′) were used to change the Gly at position 164will be mutated to Thr. An EcORI site (underlined) is also included inthe mutagenic primer as a diagnostic site.

[0169] To mutate Ala-Leu-Thr to Asn-Leu-Thr at a.a. positions 165-167,the selection primer MutKS and the mutation primer CHO165 (5′-GTG TCGTGG AAT TCA GGC AAC CTG ACC AGC GGC-3′) are used to change the Ala-165to Asn-165. An EcORI site (underlined) is included in the mutagenicprimer as a diagnostic site.

[0170] To mutate Leu-Thr-Ser to Asn-Thr-Ser at a.a. position 166-168,the selection primer MutKS and the mutation primer CHO166 (5′-TGG AACTCA GGC GCG AAT ACC AGC GGC GTG CAC-3′) were used to change the Leu-166to Asn-166. The KasI site (GGC GCC) in the original CH1 sequence ofhuman IgG1 is deliberately eliminated by changing the 3° C. into a G fordiagnostic purposes.

[0171] The phosphorylated primer pairs (selection and the respectivemutation primers) at 100 ng each are annealed to 100 ng of the stagingvector, CH1pBS, in 20 mM Tris-CH1 (pH 7.5), 10 mM MgCl₂, 50 mM NaCl in afinal volume of 20 μl by incubation at 95° C. for 3 min, and thenchilling on ice for 5 min. To the mixture, 2 to 4 units of T4 DNApolymerase, 4 to 6 units of T4 DNA ligase together with 3 l of10×synthesis buffer (CLONTECH, Palo Alto, Calif.) are added. After anincubation period of 2 hr at 37° C., the polymerization and ligationreactions are terminated by heating at 65° C. for 5 min in the presenceof 3 l of prewarmed stop solution (0.25% SDS, 5 mM EDTA). DNA from themixture is used to transform electrocompetent E. coli cells, BMH71-18mutS (repair deficient), by the method of electroporation. Transformantsare then pooled and grown overnight in SOC (20 mg/ml bacto-tryptone, 5mg/ml bacto-yeast extract, 8.6 mM NaCl, 2.5 mM KCl, 20 mM glucose) with50 g/ml ampicillin at 37° C. Mini-plasmid DNA preparations from thepooled transformants are digested with HindIII to linearize DNA notmutated with the selection primer. After the enzymes are removed byphenol extraction, the DNA is used for a second transformation withcompetent DH5 cells. Plasmid DNA that fails to be digested with HindIIIis examined for the presence of the EcORI diagnostic site (in the caseof Gly to Thr, and Ala to Asn mutations), or the absence of the KasIdiagnostic site (in the case of the Leu to Asn mutation). Finalconfirmation of the mutation is accomplished by Sanger's dideoxysequencing (Sanger et al., 1977). The CH1 region confirmed to have thedesired mutations is then excised with BamHI/BstXI enzymes and clonedinto the corresponding site of the final heavy chain expression vectorsfor hLL2, hLL2pG1g.

[0172] (3) Expression Vector for Gene Amplification.

[0173] In order to facilitate down stream process of antibodyproduction, it is desirable to utilize a gene amplification system forantibody expression. After an antibody variant is proved to haveindustrial potential, high level production could be achieved by geneamplification. From this consideration, we planned to construct theseN-linked glycosylation site mutants in the hLL2pdHL2 high levelexpression vector, a dhfr mini gene based amplification system. Heavychain mutations, HCN3, HCN4, and HCN5, were subcloned into this vectorfor expression.

[0174] The final expression constructs for these mutations weredesignated as hLL2HCN3pdHL2, hLL2HCN4 and hLL2HCN5pdHL2, respectively.

[0175] 3. Expression of mutant hLL2 and glycosylation at engineeredsites. The constant domains containing the engineered glycosylationsites were ligated to the respective variable (V) regions of hLL2. Thedifferent glycosylation mutants were expressed in murine SP2/0 myelomacells which were transfected with the heavy and light chain expressionvectors by electroporation. The engineered antibodies were purified fromthe culture supernatant of the stable antibody-producing cells byprotein A columns, and the purified proteins analyzed on SDS-PAGE underreducing conditions. The heavy chains of the glycosylation mutantsmigrated at different rates compared to that of the control antibody,hLL2, whose CH1 domain did not contain any potential glycosylationsites. Since the SDS-PAGE migration rate is inversely proportional tothe molecular sizes of the engineered oligosaccharides, the extent ofglycosylation at the different sites should be in the order ofHCN5>HCN1>HCN3>HCN2>HCN4 with hLL2HCN5 and hLL2HCN1 being the two mosthighly glycosylated Ab. By contrast, judging from the lack of migrationretardation in the light chains for the mutants KCN1-4 we concluded thatthese CK-associated sites were either not glycosylated at all, orglycosylated at an insignificant level.

[0176] 4. hLL2HCN1 and hLL2HCN5 are N-glycosylated in the CH₁ domain.The antibodies hLLHCN1, hLL2HCN5 and hLL2 were treated withN-glycosidase F (PNGase F), which specifically cleaves all types ofAsn-bound glycan from peptides, and were analyzed on reducing SDS-PAGE.The higher apparent molecular masses for the heavy chains of hLL2HCN1and hLL2HCN5 were reduced to that of hLL2 after PNGase F digestionindicating that the size difference between these Abs were attributed tothe heavy chain associated N-linked CHOs. It should be noted that, allhuman IgG₁, Abs are naturally glycosylated in the CH₂ domain at Asn297.The size differences observed might be due to differential glycosylationat the CH2 site, rather than at the engineered sites, as a result ofvariations in the culture condition. We therefore prepared F(ab′)₂fragments of hLL2HCN1, hLL2HCN5 and hLL2, and analyzed these fragmentson reducing SDS-PAGE. The size differences between the Abs were shown tobe associated with the Fd fragments (VH-CH₁), which are devoid of the Fcportion and the appended oligosaccharides, the molecular size for Fdfragments of hLL2HCN5 being larger than that of hLL2HCN1. When fragmentswere deglycosylated by PNGcase F treatment, these size differences wereeliminated and all Fd fragments migrated at the same position as theunglycosylated hLL2, suggesting that the engineered sites were actuallyused for glycosylation and the extent of glycosylation for HCN5 site waslarger than that of HCN1.

[0177] The N-linked oligosaccharide moieties in the CH₁, domain ofhLL2HCN1 were directly visualized by CHO-specific labeling. Theoligosaccharide moieties attached to the were first periodate oxidized.The aldehydes groups generated were then covalently conjugated withbiotin, which was probed and visualized by streptavidin-peroxidase in awestern blotting analysis. As we anticipated, only the heavy chain butnot light chains of both hLL2 and hLL2HCN1 were visible with CHOlabeling. When quantified with densitometry, the intensity of labeledCHOs in hLL2HCN1 was approximately 2.5-fold of that in hLL2. The proteincontents of the different Abs analyzed were comparable, as shown bycoomassie blue-stained SDS-PAGE. We attributed this difference inintensity to be the result of additional glycosylation in the engineeredHCN1 site. This was confirmed when the F(ab′)₂ fragments were subjectedto the same analysis: only the Fd fragment of hLL2HCN1 but not that ofhLL2 demonstrated CHO specific labeling. By contrast, potential CKglycosylation sites were not found to be glycosylated.

[0178] It should be noted that, unlike the VK-appended glycosylationsite which exhibited heterogeneity in the degree of glycosylation, onlyone discrete band was observed in the SDS-PAGE analysis for hLL2(HCN1)Fd fragment. It is speculated that almost all of the Fd fragments ofhLL2(HCN1) were glycosylated, and the degree of glycosylation wasrelatively homogenous, a desirable property that would facilitate theirsubsequent characterizations and applications.

[0179] 5. WN competitive binding assay. The antigen-binding property ofthese two antibodies was evaluated by competition binding with mLL2 toan LL2 anti-idiotype antibody (WN). This assay showed that the bindingactivity of hLL2HCN1 and hLL2HCN2 to WN is indistinguishable from thatof hLL2. (FIG. 11).

EXAMPLE 12

[0180] Site-Specific Conjugation of Aminobenzyl DTPA andDextran-Doxorubicin to hLL2HCN1 and hLL2HCN5.

[0181] The site-specific modification of the F(ab′)₂ fragments ofantibodies with DTPA was as described. See Leung et al., J. Immunol.154:5919 (1995). F(ab′)₂ fragment (˜1 mg/ml) was oxidized with 15 mM ofsodium metaperiodate at 4° C. for 1 h. The oxidized material waspurified, mixed with 545-fold molar excess of aminobenzyl DTPA and thepH was adjusted to 5.97. The mixture was incubated in the dark atambient temperature for 5 h, and then kept at 4° C. for 18 h. Theconjugates were stabilized with 1O mM of sodium cyanoborohydride,purified and concentrated. The chelator:F(ab′)₂ ratio was determined bymetal binding assays and use of indium acetate spiked with ¹¹¹In. SeeMeares et al., Anal. Biochem. 142:68 (1984). Radiolabeling was performedas described. See Leung et al., (1995), supra. The number of DTPAmolecules conjugated to F(ab′)₂ fragment was determined by metal-bindingassay using In/In-111 system. Briefly, 40 μg of the conjugates wasincubated for 30 min with a known excess of indium acetate, spiked withIn-111 acetate. The solution was made 10 mM in EDTA, and incubated forfurther 10 min. The labeling was analyzed by ITLC using 10 mM EDTA fordevelopment. DOX-dextran conjugate was prepared as described by Shih etal., Cancer Res. 51: 4192 (1991), using amino-dextran of 18 kDa as theintermediate carrier. The intermediate conjugate possessed asubstitution level of 10.5 DOX molecules per dextran polymer.DOX-dextran was then conjugated with the F(ab′)₂ fragment of hLL2HCN1 orhLL2HCN5. Briefly, the antibody fragment was concentrated to 1O mg/ml in0.1 M sodium acetate buffer, pH 5.5, and treated with 20 mM of sodiummetaperiodate in the dark at 4° C. for 60 min. The oxidized antibody waspurified on a Bio-Spin column (Bio-Rad) that was pre-equilibrated in0.05 M HEPES buffer, pH 8.0, containing 0.1 M NaCl, and then treatedwith DOX-dextran (4 equivalents) at room temperature for 24 h. Aftersodium borohydride reduction, the conjugated product was purified on aBio-gel A-0.5 m gel column (Bio-Rad). The protein fractions were pooledand concentrated in Centricon 50 concentrator (Amicon, Beverly, Mass.).The trace amount of intermediates in the protein conjugates was removedby repetitive washing with the conjugation buffer as evaluated by HPLCon Bio-Sil Sec size exclusion column (Bio-Rad).

EXAMPLE 13

[0182] CH₁-Appended Oligosaccharides can be Used as EfficientConjugation Sites for Chelates and/or Drugs.

[0183] Under mild chemical conditions, an average of 1.6 and 2.97molecules of DTPA were conjugated onto each F(ab′)₂ fragment of hLL2HCN1and hLL2HCN5, respectively (Table 2). Both conjugates demonstrated highefficiencies in ¹¹¹In incorporation (92% for hLL2HCN1, 91% forhLL2HCN5). No significant changes in immunoreactivities were observedbefore and after DTPA conjugation of the glycosylation mutant fragments,as evaluated in a WN competitive blocking assay. HCN5-appended CHOappeared to be more reactive for chelate conjugation when compared tothe HCN1-appended CHO; almost twice as many DTPA molecules could beincorporated into the HCN5 site.

[0184] Leung et al. (1995), supra, has shown that the VK-appended CHOfound in murine LL2 can be used as a site-specific conjugation site forsmall chelates without reducing the Ag binding property of the Ab. Theeffect of conjugating this VK-appended CHO with dextran-DOX complex onimmunoreactivity was examined. The dextran-DOX complex was generated bychemically incorporating an average of 10 DOX molecules onto an 18 kDaamino-dextran polymer. Using the amino-dextran as the carrier for DOX,approximately 5.1 DOX molecules on average were incorporated onto theVK-appended CHO of murine LL2, and a reduction of close to 60% ofimmunoreactivity as evaluated by cell binding and ELISA assays, wasobserved. See Table 3. Conjugation of slightly higher number of DOXmolecules (6.8) onto the HCN1 CHO, however, was comparatively lessdetrimental in term of its effect on immunoreactivity; only 30%reduction in the resultant binding affinity was noted. In contrast, nosignificant changes in Ag binding property (less than 5% reduction) wereapparent when similar number of DOX molecules (7.2) was conjugated atthe HCN5 CHO. See Table 3.

[0185] The molecular masses of the F(ab′)₂ fragments of hLL2, hLL2HCN1and hLL2HCN5 determined by mass spectrometry analysis (Mass Consortium,San Diego, Calif.) were 99,000, 102,400 and 103,800, respectively sincethese fragments are identical in sequences, except at the engineeredsite (one amino acid difference), and the fragments did not carry theglycosylated Fc portion, the molecular mass difference between theF(ab′)₂ of hLL2 and the glycosylation mutant should represent themolecular weights of the different CH1-appended CHOs, i.e., 3.4 and 4.8kD for the CHOs at the HCN1 and the HCN5 sites, respectively.

[0186] By PNGase F digestion, the CH1-appended CHOs of hLL2HCN1 andhLL2HCN5 were released for profiling and sequencing analyses usingfluoropore-assisted carbohydrate electrophoresis (FACE). Heterogenouspopulations of CH1-appended CHO species were identified. About 60% ofthe oligosaccharides from HCN5 site were of the larger tri-antennarystructure, while that from HCN1 were mainly bi-antennary (>90%). Theseresults are consistent with the mass spectrometry studies indicating alarger average molecular size of the CHO at the HCN5 sites compared tothat of HCN1.

[0187] It should be emphasized that the above-described examples merelydescribe several specific embodiments of the invention, and applicantsdo not intend to be limited as to scope of claims by these specificexamples. Applicants also incorporate by reference all publications andpatents cited in the specification. TABLE 2 Site-specific conjugation ofDTPA and radiolabeling. Antibody (%) Efficiency^(a) ¹¹¹In labelingImmunoreactivity F(ab′)₂ DTPA DTPA//F(ab′)₂ % Incorp.^(b) μCi/μg^(c)ID₅₀ % of hLL2^(d) hLL2 Non-conj. NA NA NA 0.384 100 (±0.021) hLL2HCN1Non-conj. NA NA NA 0.355 100 (±0.038) Conjugated 1.6 92 6 0.387 100.8(±0.042) hLL2HCN5 Non-conj. NA NA NA 0.443 115.4 (±0.039) Conjugated2.97 91 5.6 0.356  92.7 (±0.077)

[0188] TABLE 3 Site-specific conjugation of doxorubicin.Immunoreactivity Antibody (%) F(ab′)₂ Yield^(a) (DOX/ Cell ELISA^(d)Dextran-DOX (%) Efficiency^(b) F(ab′)₂₎ binding^(c) mLL2 Non-conj. NA NA100 100 Conjugated 55 5.1  41.9  42.2 hLL2HCN1 Non-conj. NA NA 100 100Conjugated 30 6.8  70  70.6 hLL2HCN5 Non-conj. NA NA ND 100 Conjugated80 7.2 ND  94.8

We claim:
 1. A method of producing an antibody or antigen-bindingfragment thereof comprising: expressing said antibody or antigen-bindingfragment thereof which is engineered to contain a glycosylation site inthe non-Fc constant heavy chain region, wherein said antibody orantigen-binding fragment is glycosylated in the CH1 region, or in theconstant light chain region, wherein genes encoding said heavy chain andlight chain regions have been engineered with a mutation such that aglycosylation site is created in the CH1 region gene or the constantlight chain gene, and operably linked to expression control elements inan expression vector, in a cell that allows glycosylation; and producingsaid antibody or antibody fragment glycosylated in the CH1 region or thelight chain constant region in said cell.
 2. The method of claim 1,wherein said expression vector comprises an amplifiable dihydrofolatereductase (dhfr) gene.
 3. The method of claim 2, wherein said expressionvector is pdHL2.
 4. The method of claim 3, wherein said cell is a SP2/0myeloma cell.
 5. The method of claim 1, wherein the antibody or fragmentthereof comprises a humanized antibody or antigen-binding fragmentthereof.
 6. The method of claim 1, wherein the antibody or fragmentthereof comprises a humanized B-cell specific antibody orantigen-binding fragment thereof.
 7. The method of claim 6, wherein saidglycosylation is located on a site selected from the group consisting ofthe HCN1, HCN2, HCN3, HCN4, and HCN5 sites (SEQ ID NOS: 10-14) of FIG.12.
 8. The method of claim 7, wherein said glycosylation site is theHCN5 site (SEQ ID NO: 10) of FIG.
 12. 9. The method of claim 7, whereinsaid glycosylation site is the HCN1 site (SEQ ID NO: 10) of FIG.
 12. 10.The method of claim 6, wherein the antibody or antigen-binding fragmentthereof is engineered to contain a glycosylation site is an antibody orantigen-binding fragment thereof having the binding specificity of thehLL2 antibody.
 11. The method of claim 1, wherein said glycosylation islocated at a N-linked glycosylation site.
 12. The method of claim 10,wherein said expression vector comprises an amplifiable dihydrofolatereductase (dhfr) gene.
 13. The method of claim 12, wherein saidexpression vector is pdHL2.
 14. The method of claim 13, wherein saidcell is a SP2/0 myeloma cell.
 15. The method of claim 1, wherein saidantibody or fragment thereof is encoded by a DNA molecule comprising aDNA sequence comprising an engineered glycosylation site in the DNAsequence encoding the CH1 region or the constant light chain region.